Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Development of a Fluorescence-based Trypanosoma cruzi CYP51 Inhibition Assay for Effective Compound Triaging in Drug Discovery Programmes for Chagas Disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51.